Escherichia coli strain with enhanced l-threonine productivity and method of producing l-threonine using the same

ABSTRACT

An L-threonine-producing  Escherichia coli  in which a promoter of a phosphoenolpyruvate carboxylase (ppc) gene on the chromosome is substituted with a promoter of a cysteine synthase (cysK) gene and a method of producing L-threonine by using the same are disclosed. The recombinant  Escherichia coli  may produce L-threonine in a high yield, and thus may be widely used in medical, pharmaceutical, and feed industries, particularly for an animal feed.

TECHNICAL FIELD

One or more embodiments of the present invention relate to anL-threonine-producing Escherichia coli (E. coli) strain with enhancedL-threonine productivity in which a promoter of a phosphoenolpyruvatecarboxylase (ppc) gene on the chromosome is substituted with a promoterof a cysteine synthase (cysK) gene, and a method of producingL-threonine using the same.

BACKGROUND ART

L-threonine is an essential amino acid and is widely used as an animalfeed additive or food additive, and is also used as a raw material formedical fluid or drug synthesis. While animal protein contains asufficient amount of L-threonine, vegetable protein is deficient inL-threonine. Thus, L-threonine is likely to be deficient in animalsmainly on vegetarian diets, and thus in particular it is widely used asan additive for animal feed.

L-threonine is produced mainly by a fermentation process usingEscherichia coli (E. coli) or Corynebacterium, which is developed byartificial mutagenesis or genetic recombination. To produce L-threonine,a mutant strain derived from a wild-type strain of Escherichia coli (E.coli), Corynebacteria sp., Serratia sp., or Providencia sp is used.Examples of the mutant strain include an amino acid analogue- ordrug-resistant mutant strain, and a diaminopimellic acid, a methionine,a lysine, or an isoleucine auxotrophic mutant strain that has also anamino acid analogue- or drug-resistance. Among methods of producingL-threonine using a mutant strain, a method of using a microorganismthat belongs to Escherichia coli species, has diaminopimellic acid andmethionine auxotroph phenotypes, and is mutated so that biosynthesis ofL-threonine is not affected by feedback inhibition of threonine isdisclosed in Japanese Patent No. 10037/81.

A fermentation process using a recombinant strain can also be used inproduction of L-threonine. For example, Japanese Patent ApplicationPublication No. 05-227977 discloses a method of producing L-threonineusing a recombinant E. coli into which a threonine operon consisting ofgenes encoding aspartokinase, homoserine dehydrogenase, homoserinekinase, and threonine synthase is introduced in a plasmid form, and amethod of producing L-threonine using threonine-producing Brevibacteriumflavum into which a threonine operon derived from E. coli is introduced(TURBA E, et al, Agric. Biol. Chem. 53:2269-2271, 1989).

In general, the expression of a specific gene in a microorganism may beenhanced by increasing the copy number of the gene in the microorganism.For this, a plasmid that gives a greater copy number to a microorganismis used [Sambrook et al., Molecular Cloning, 2th, 1989, 1.3-1.5]. Thatis, the number of the gene may be increased by as many as the copynumber of the plasmid per a single microorganism by inserting a targetgene into the plasmid whose copy number may be maintained at a highlevel and then transforming the microorganism with the obtainedrecombinant plasmid. Attempts have also been made to enhance theproductivity of threonine using this method and a partial success wasreported (U.S. Pat. No. 5,538,873). However, this technology using aplasmid induces excessive expression of only a specific gene in mostcases, thereby imposing a heavy burden on a host microorganism.Furthermore, plasmids may be lost during culturing of a recombinantstrain, thereby decreasing plasmid stability. To address these problemsof the method of producing threonine by using a recombinant strain intowhich a plasmid is introduced, addition of an antibiotic into a cultureand methods of using a plasmid whose expression is controllable havebeen developed_ [Sambrook et al. Molecular Cloning, 2th, 1989, 1.5-1.6,1.9-1.11]. In the case of using the plasmid whose expression iscontrollable, to alleviate the burden on a host microorganism and obtaina large amount of cells, during the growth phase, a host microorganismis cultured under conditions where the expression of a target gene onthe plasmid does not occur, and after the sufficient growth of the hostmicroorganism, temporary expression of the gene is induced, therebyobtaining a target material. However, methods using plasmids whoseexpression is controllable can be used only when a final gene product isa protein or a secondary metabolite. In a case where a gene product is aprimary metabolite that is produced at the same time when microorganismsbegin to grow, expression of a target gene must be induced during thegrowth phase. Otherwise, it is difficult to anticipate the accumulationof the primary metabolite. Since threonine belongs to a primarymetabolite, the latter case is also applied to threonine.

Thus, to enhance the productivity of threonine, which is a primarymetabolite, inserting genes involved in threonine biosynthesis intochromosomal DNA of a microorganism is disclosed in U.S. Pat. No.5,939,307, instead of using a method of introducing a plasmid withthreonine biosynthesis-related genes into a microorganism. Methods ofincreasing the threonine biosynthesis-related genes and the expressionthereof have been diversely developed, but there is still a need fordeveloping a method of more economically producing L-threonine in a highyield.

To increase the production yield of L-threonine, research on abiosynthesis pathway from oxaloacetate to threonine has been intensivelyconducted. With regards to this, we intended to first induce the flow ofcarbon along a pathway from phosphoenolpyruvate to oxaloacetate byenhancing the activity of phosphoenolpyruvate carboxylase involved in astep right before the biosynthesis of L-threonine. For this, we studiedand found that a microorganism strain capable of producing L-threoninein which a promoter of a phosphoenolpyruvate carboxylase (ppc) gene onthe chromosome was substituted with a promoter of a gene encodingcysteine synthase (cysK) so as to increase the expression of a geneencoding ppc, which is a first enzyme in the biosynthesis of L-threonineafter glycolysis, produced L-threonine in a high yield, thus completingthe present invention.

DISCLOSURE OF INVENTION Technical Problem

One or more embodiments of the present invention provide an Escherichiacoli (E. coli) strain capable of producing L-threonine in high yield.

One or more embodiments of the present invention also provide a methodof producing L-threonine using the E. coli strain, whereby L-threoninemay be efficiently produced.

Technical Solution

The present invention provides an L-threonine-producing microorganism inwhich a promoter of a natural phosphoenolpyruvate carboxylase (ppc) geneon the chromosome is substituted with a promoter of a cysteine synthase(cysK) gene.

In the L-threonine-producing microorganism, the expression of ppcincreases in such a way that a promoter of a gene encoding ppc, whichconverts phosphoenolpyruvate produced after glycolysis to oxaloacetate,the starting material of L-threonine biosynthesis, is substituted withthe promoter of the cysK gene, and the productivity of L-threonine mayincrease, accordingly.

The microorganism may include a prokaryotic or eukaryotic cell capableof producing L-threonine in which a promoter of a ppc gene on thechromosome is substituted with a promoter of a cysK gene. For example,the microorganism may be a microorganism strain belonging to Escherichiagenus, Erwinia genus, Serratia genus, Providencia genus, Corynebacteriumgenus, or Brevibacterium genus. In particular, the microorganism may bea microorganism belonging to Enterobacteriaceae family, and moreparticularly, a microorganism belonging to Escherichia genus. Mostparticularly, the microorganism may be Escherichia coli CA030031 (KCCM10910).

The E. coli CA030031 is derived from E. coli KCCM 10541 which is derivedfrom a L-threonine-producing E. coli, KFCC 10718 (Korean PatentPublication No. 92-8395). The E. coli KFCC 10718 has a resistance to anL-methionine analogue, a methionine auxotroph phenotype, a resistance toan L-threonine analogue, a leaky isoleucine auxotroph phenotype, aresistance to an L-lysine analogue, and a resistance to α-aminobutyricacid, and is capable of producing L-threonine. Thus, the microorganismmay also include a mutant microorganism for producing L-threonine, inaddition to a wild-type microorganism. For example, the mutantmicroorganism may be a microorganism that has a resistance to anL-methionine analogue, a methionine auxotroph phenotype, a resistance toan L-threonine analogue, a leaky isoleucine auxotroph phenotype, aresistance to an L-lysine analogue, and a resistance to α-aminobutyricacid, and belongs to E. coli capable of producing L-threonine.

In an embodiment, the microorganism may be E. coli that has a methionineauxotroph phenotype and resistances to a threonine analogue, a lysineanalogue, an isoleucine analogue and a methionine analogue. For example,the L-methionine analogue may be at least one compound selected from thegroup consisting of D,L-methionine, norleucine, α-methylmethionine, andL-methionine-D,L-sulfoximine, the L-threonine analogue may include atleast one compound selected from the group consisting ofα-amino-β-hydroxy valeric acid and D,L-threonine hydroxamate, and theL-lysine analogue may be at least one compound selected from the groupconsisting S-(2-aminoethyl)-L-cysteine and δ-methyl-L-lysine. Examplesof the mutant microorganism may include a microorganism in which a pckAgene involved in converting oxaloacetate (OAA) into phosphoenol pyruvate(PEP), which is an intermediate involved in the biosynthesis ofL-threonine, is inactivated, a microorganism in which a tyrR generepressing a lysC gene which is involved in conversion of oxaloacetateinto aspartate is inactivated, or a microorganism in which a galR generepressing the expression of a galP gene which is involved in the influxof glucose is inactivated.

In the microorganism of the present invention, the promoter of the ppcgene on the chromosome is substituted with the promoter of the cysK geneso as to increase the expression thereof. The promoter of the cysK geneused herein may be derived from a cysK gene with a high expression rate,and may have a nucleotide sequence of SEQ ID NO: 10.

The present invention also provides a method of producing L-threonine,the method including: culturing a transformed microorganism withenhanced L-threonine productivity in which a promoter of a natural ppcgene on the chromosome is substituted with a promoter of a cysK gene;and isolating L-threonine from the culture of the microorganism.

In the method of producing L-threonine, the transformed microorganismmay be E. coli, for example, E. coli CA030031 (KCCM 10910).

One or more embodiments of the present invention will now be describedmore fully with reference to the following examples. However, theseexamples are provided only for illustrative purposes and are notintended to limit the scope of the present invention.

ADVANTAGEOUS EFFECTS

As described above, according to the present invention, in amicroorganism in which a promoter of a ppc gene on the chromosome issubstituted with a promoter of a cysK gene, the expression of the ppcgene, which is an enzyme converting phosphoenolpyruvate to oxaloacetatethat is a precursor of L-threonine biosynthesis, increases, therebysignificantly enhancing the productivity of L-threonine by 16% orhigher. The microorganism may produce L-threonine in a high yield, andthus may be widely used in medical, pharmaceutical, and feed industries,particularly for an animal feed.

DESCRIPTIONS OF DRAWINGS

FIG. 1 is a diagram illustrating a process of constructing a recombinantvector pUC-PcysK; and

FIG. 2 is a diagram illustrating a process of constructing a recombinantvector pUCpcysKmloxP.

MADE FOR INVENTION Example 1 Construction of Recombinant VectorpUCpcysKmloxP

Preparation of Pcysk Fragment

To obtain 0.3 kb DNA fragment containing a promoter of a cysK gene (SEQID NO: 10), the genomic DNA (gDNA) of W3110, which is E. coli wild typestrain, was extracted using a QIAGEN Genomic-tip system, and apolymerase chain reaction (PCR) was performed using the gDNA as atemplate and a PCR HL premix kit (manufactured by BIONEER, Korea). Toamplify the promoter of the cysK gene, the PCR was performed usingprimers of SEQ ID NOS: 1 and 2 as follows: 30 cycles of denaturation at94° C. for 30 seconds, annealing at 55° C. for 30 seconds and elongationat 72° C. for 2.5 minutes.

The PCR products were digested with KpnI and EcoRV, electrophoresized ona 0.8% agarose gel, and then eluted to obtain 0.3 Kb DNA fragment(hereinafter, referred to as “PcysK fragment”).

(2) Construction of Recombinant Vector pUC-PcysK

FIG. 1 is a diagram illustrating a process of constructing recombinantvector pUC-PcysK containing a promoter of a cysK gene.

Plasmid pUC19 (New England Biolabs, USA) and the PcysK fragment obtainedaccording to Example 1-(1) were each digested with restriction enzymesKpnI and SmaI, and ligated with each other to construct vectorpUC-PcysK.

(3) Construction of Recombinant Vector pUCpcysKmloxP

FIG. 2 is a diagram illustrating a process of constructing a recombinantvector pUCpcysKmloxP.

In general, in an experiment of gene deletion caused by one-stepinactivation, whenever one gene is deleted, one sequence of arecombinase recognition site loxP remains on a chromosomal DNA. It hasbeen reported that due to the sequences of loxP remaining on thechromosomal DNA, when the strains are additionally modified for furtherdevelopment, the efficiency may be significantly decreased (Nagy A.,Genesis, 26:99, 2000). An improved method of gene deletion using loxPmutants, which are named lox71 and lox 66 has been proposed by Suzuki(Appl. Environ. Microbiol. 71:8472, 2005). Thus, to more efficientlysubstitute a promoter of a ppc gene on the chromosome with a promoter ofa cysK gene by using the loxP mutants, we constructed vectorpUCpcysKmloxP having both a mutant loxP-Cm^(R)-loxP cassette and thepromoter of the cysK gene.

As shown in FIG. 2, the PCR was performed using plasmid pACYC184 (NewEngland Biolab) as a template by using primers of SEQ ID NOS: 3 and 4 asfollows: 30 cycles of denaturation at 94° C. for 30 seconds, annealingat 55° C. for 30 seconds and elongation at 72° C. for 1 minute to obtain1.1 kb of PCR fragment. The vector pUC-PcysK constructed according toExample 1-(2) and 1.1 kb of DNA fragment obtained using pACYA184 as atemplate were each digested with restriction enzymes NdeI/KpnI, ligatedwith each other, and transformed into E. coli. Then, cell having DNAaccurately ligated with the vector were selected using a general method,and plasmid pUCpcysKmloxP was purified from the culture of the cells.

Example 2 Preparation of Recombinant E. coli KCCM 10541-PcyK-ppc

To substitute a native promoter of a ppc gene (SEQ ID NO: 9) encodingphosphoenolpyruvate carboxylase on the chromosome with a promoter of acysK gene, a known one-step inactivation method (Warner et al., PNAS, 6;97(12):6640, 2000) was performed on E. coli KCCM 10541.

First, PCR was performed using the plasmid pUCpcysKmloxP constructedaccording to Example 1 as a template by using primers of SEQ ID NOS: 5and 8 as follows: 30 cycles of denaturation at 94° C. for 30 seconds,annealing at 55° C. for 30 seconds and elongation at 72° C. for 1 minuteto obtain DNA fragments and the obtained DNA fragments were purifiedusing a QIAGEN kit (PCR Purification kit). Subsequently, PCR was furtherperformed using primers of SEQ ID NOS: 6 and 7 and the purified DNAfragments as a template as follows: 30 cycles of denaturation at 94° C.for 30 seconds, annealing at 55° C. for 30 seconds and elongation at 72°C. for 1 minute. The resulting DNA fragments were purified, the purifiedDNA fragments were introduced by electroporation into E. coli KCCM 10541into which vector pKD46 was introduced (Proc. Natl. Acad. Sci. U.S.A.97(12), 6640-6645(2000)), and a single colony was selected on aLuria-Bertani (LB) plate containing 15 μg/mL of chloramphenicol. Theselected strain was a strain in which the DNA fragment was inserted intoa promoter site of a ppc gene. Vector pJW168 (BMG Biothechnol. 2001;1:7. Epub 2001 Sep. 26) was introduced into the selected strain toprepare recombinant E. coli KCCM 10541-PcyK-ppc in which the naivepromoter of the ppc gene was substituted with the promoter of the cysKgene, by removing antibiotics-resistance gene.

Example 3 Comparison in L-threonine Productivity Between RecombinantMicroorganisms

The recombinant microorganism prepared according to Example 2 wascultured in a threonine titer medium, as shown in Table 1 below, in anErlenmeyer flask, and it was confirmed whether the productivity ofL-threonine was improved.

TABLE 1 Composition Concentration (per liter) Glucose 70 g KH₂PO₄ 2 g(NH₄)₂SO₄ 25 g MgSO₄•7H₂O 1 g FeSO₄•7H₂O 5 mg MnSO₄•4H₂O 5 mgDL-methionine 0.15 g Yeast extract 2 g Calcium carbonate 30 g pH 6.8

1 platinum loop of E. coli KCCM 10541 and E. coli KCCM 10541-PcysK-ppcthat were cultured in a LB solid medium in an incubator at 33° C.overnight were each inoculated in 25 ml of titer medium, as shown inTable 1, and cultured in the incubator at 33° C. at 200 rpm for 48hours. The results are shown in Table 2 below.

As shown in Table 2, when the parent strain E. coli KCCM 10541 wascultured for 48 hours, it produced 29.8 g/L of L-threonine, while the E.coli KCCM 10541-PcysK-ppc of Example 2 produced 34.7 g/L of L-threonine,which has a 4.9 g/L higher productivity than that of the parent strain.Thus, it was confirmed that the recombinant strain in which the promoterof the ppc gene was substituted with the promoter of the cysK gene hasenhanced L-threonine productivity. The transformed E. coli KCCM10541-PcysK-ppc was named E. coli CA030031 and deposited in KoreanCulture Center of Microorganisms (KCCM) on Dec. 20, 2007 (Accession No:KCCM 10910).

TABLE 2 Strain L-threonine (g/L) KCCM10541 (parent strain) 29.8KCCM10541-PcysK-ppc 34.7

While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

1. An L-threonine-producing Escherichia coli strain in which a promoterof a phosphoenolpyruvate carboxylase (ppc) gene on the chromosome issubstituted with a promoter of a cysteine synthase (cysK) gene.
 2. TheEscherichia coli strain of claim 1, having a methionine auxotrophphenotype, and resistances to a threonine analogue, a lysine analogue,an isoleucine analogue and a methionine analogue.
 3. The Escherichiacoli strain of claim 1, being Escherichia coli CA030031 (Accession No:KCCM 10910).
 4. A method of producing L-threonine, the methodcomprising: culturing the Escherichia coli strain according to claim 1;and isolating L-threonine from the culture of the Escherichia colistrain.
 5. A method of producing L-threonine, the method comprising:culturing the Escherichia coli strain according to claim 2; andisolating L-threonine from the culture of the Escherichia coli strain.6. A method of producing L-threonine, the method comprising: culturingthe Escherichia coli strain according to claim 3; and isolatingL-threonine from the culture of the Escherichia coli strain.